Banana plant named ‘QCAV-4’

ABSTRACT

A new banana cultivar ‘QCAV-4’ is provided that, when under significant disease pressure, remains largely free from infection by Fusarium wilt tropical race 4 (TR4). In the absence of such significant disease pressure, ‘QCAV-4’ appears to be essentially phenotypically identical to the wild type parent Cavendish Grand Nain. This includes in relation to immature and mature plant characteristics, fruit characteristics, and yield.

Latin name of the genus and species of the plant claimed: Musaacuminata.

Variety denomination: ‘QCAV-4’.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinct cultivar of bananaplant named ‘QCAV-4’. The new plant resulted from transformation ofparent Cavendish Grand Nain (unpatented) by T-DNA insertion andselection. A resulting transgenic plant named ‘QCAV-4’ was selected whengrowing in a cultivated area in Lambells Lagoon, Northern Territory,Australia.

BRIEF SUMMARY OF THE INVENTION

‘QCAV-4’ is a transgenic cultivar produced from Cavendish Grand Nain.For initial transformation, embryogenic cell suspensions (ECS) weregenerated from immature male flowers from the bell (flower) of CavendishGrand Nain. The bells were collected in North Queensland, Australia andindexed for virus infection. The ECS were transformed usingAgrobacterium mediated transformation. The transformation cassetteincluded a selectable marker gene, neomycin phosphotransferase (NPT II).The resistance gene was a gene isolated from Musa acuminata subsp.malaccensis which is resistant to Fusarium wilt tropical race 4 (TR4).The resistance gene was under the control of the nos promoter.Potentially transformed cells were placed on kanamycin to select NPT IIresistant cells. These were then regenerated into whole plantlets andmultiplied. Presence of the transgenes were confirmed by PCR. Multipliedplantlets were transferred to a farm in Lambells Lagoon, NorthernTerritory, Australia and acclimatized in a screenhouse. These plantstogether with appropriate controls were planted into a plot whereCavendish bananas had been previously grown and had been severelyaffected by Fusarium wilt TR4. The plot was “seeded” further withpseudostem segments from infected Cavendish plants. Plants wereregularly inspected for TR4 symptoms over a three-year period. Multipleindependent transformed lines demonstrated strong resistance to TR4 ascompared to the parental Cavendish Grand Nain, which is highlysusceptible. Morphological characteristics of plants and fruit wereassessed, bunch weight was measured, and molecular analysis wasperformed. One line was selected based on morphological and molecularanalysis, and named ‘QCAV-4’.

The ‘QCAV-4’ cultivar is distinguished from other banana varieties,including the parent, by having a strong resistance phenotype toFusarium wilt tropical race 4 (TR4). It is substantially phenotypicallyidentical to its parent in the absence of disease pressure.

Asexual reproduction of ‘QCAV-4’ by tissue culture in Brisbane City,Queensland, Australia in combination with field assessment in LambellsLagoon, Northern Territory, Australia, shows that the foregoingcharacteristic resistance to Fusarium wilt TR4 reproduces true to type.

The following detailed description concerns progeny lines asexuallypropagated from the original line by tissue culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a digital image of Southern blot analysis of wild typeCavendish Grand Nain (parent) and ‘QCAV-4’, showing endogenous RGA2copies and transgenic RGA2 insertions.

FIG. 1B is a digital image of Southern blot analysis of wild typeCavendish Grand Nain (parent) and independent transgenic lines ‘RGA2-2’,‘RGA2-3’, ‘RGA2-4’ (clonal progenitor of ‘QCAV-4’), ‘RGA2-5’ and‘RGA2-7’ showing distinct pattern of transgenic RGA2 insertions, inaddition to the three endogenous RGA2 copies.

FIG. 2A is a schematic diagram showing the general organization of theinsert in event ‘QCAV-4’.

FIG. 2B is a schematic diagram showing the rearrangements in thegenome/T-DNA and inter T-DNA junctions of event ‘QCAV-4’, demonstratingseven (7) new ORFs have been identified in the inter T-DNA regions ofthe transgenic insert of ‘QCAV-4’.

FIG. 2C is a schematic diagram showing that there is no evidence ofexpression of the seven new open reading frames (ORFs) in ‘QCAV-4’.

FIGS. 3-4 are photographs showing ‘QCAV-4’ at adult stage.

FIG. 5 is photograph showing ‘QCAV-4’ at adult stage and developingfruit.

FIGS. 6-7 are photographs showing ‘QCAV-4’ at adult stage.

FIGS. 8-9 are graphical representations of the data shown in Tables 7-8,respectively.

FIG. 10 is a graphical representation of the data shown in Table 9.

The colors of an illustration of this type may vary with lighting andother conditions under which conditions and, therefore, colorcharacteristics of this new cultivar should be determined with referenceto the observations described herein, rather than from theseillustrations alone.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file,created on Dec. 14, 2021, 12 KB, which is incorporated by referenceherein. In the accompanying sequence listing:

SEQ ID NOs: 1-7 are new ORF sequences found in ‘QCAV-4’ that resultedfrom the transgenic event.

DETAILED DESCRIPTION

The following detailed description of ‘QCAV-4’ is based on observationsof plants that are approximately 25 months old. The ‘QCAV-4’ plants havebeen observed growing in a cultivated area in Lambells Lagoon, NorthernTerritory, Australia. Certain characteristics of this cultivar, such asgrowth and color, may change with changing environmental conditions(such as, light, temperature, moisture, nutrient availability, or otherfactors). Color descriptions and other terminology are used inaccordance with their ordinary dictionary descriptions, unless thecontext clearly indicates otherwise.

BOTANICAL DESCRIPTION

Scientific name: Musa acuminata ‘QCAV-4’

Parentage: Cavendish Grand Nain

Plant:

In the absence of significant disease pressure, ‘QCAV-4’ appears to beessentially phenotypically identical to the wild type parent ‘CavendishGrand Nain’ (unpatented) (Table 1). This includes in relation toimmature and mature plant characteristics, fruit characteristics, andyield.

TABLE 1 Comparison of ‘QCAV-4’ to parent ‘Cavendish Grand Nain’*‘Cavendish Organ/Plant Part: Context ‘QCAV-4’ Grand Nain’ Ploidy:triploid triploid Pseudostem: overlapping of weak weak leaf sheathsPseudostem: tapering absent or weak absent or weak Pseudostem: colourpurple purple Pseudostem: anthocyanin medium to strong mediumcolouration Pseudostem: colour of inner purple purple side of basalsheath Plant: compactness of crown compact compact Plant: growth habitdrooping drooping Petiole: attitude of wings at base curved outwardscurved outwards Leaf blade: colour of midrib on green green lower sideLeaf blade: shape of base both sides acute both sides acute Leaf blade:waxiness on lower medium weak to medium side Leaf blade: width broadbroad Leaf blade: glossiness of upper absent absent side Peduncle:diameter large large Peduncle: pubescence present present Peduncle:curvature medium to strong medium to strong Bunch: length long longBunch: shape cylindrical cylindrical Bunch: attitude of fruitsmoderately moderately turned up turned up Bunch: compactness mediummedium Bunch: number of hands many many Rachis: attitude of male partvertical vertical Rachis: prominence of scars weak weak Rachis:persistence of bracts absent or weak absent or weak Rachis: persistenceof present present hermaphrodite flowers Fruit: colour of peel (beforegreenish yellow greenish yellow maturity) (RHS 141C) (RHS 141C) Fruit:persistence of floral organs present present Male inflorescence:persistence present present Male inflorescence: shape narrow ovatenarrow ovate Male inflorescence: opening closed or slightly closed orslightly of bracts open open Bract: colour of inner side orange redorange red Bract: shape of apex broad acute broad acute *For mostcharacteristics, 4-5 individual plants were assessed.

However, a clear phenotype is observable under pressure from Fusariumwilt tropical race 4 (TR4).

In March 2018, an expanded field trial was planted which included 50replicates of each of the four events from Trial 1, in 10×5 randomizedplot design. In addition to recording disease incidence, detailedagronomic information such as bunch weight, number of fingers on the tophand and crop cycling time is also collected. Since the trial began,agronomic data for the plant crop and at least two ratoon crops werecollected. The trial is ongoing. Based on the results of these fieldtrials and molecular characterisation, ‘QCAV-4’ was selected.

The disease status of plants is assessed by the presence ofcharacteristic disease symptoms (both external and internal) and bymolecular testing of vascular tissue for the presence of the fungalpathogen TR4. The plants are inspected on a weekly basis and plantsshowing the characteristic external symptoms of the disease identified.About 1-2 weeks later, the pseudostem of these plants is cut andexamined for the presence of the highly characteristic internal vasculardiscolouration associated with TR4 infection. DNA is extracted from theinfected vascular tissue, and a highly sensitive PCR test is used todetect the presence of TR4, and this is confirmed by sequencing. The TR4fungus from discoloured vascular tissue is obtained and DNA extractedand analysed using PCR to confirm the presence of TR4 (and also bysequencing).

As shown in Table 2, ‘QCAV-4’ can remain largely disease-free under thesame conditions of TR4 pressure leading to greater than 80% infectionrates in wild type ‘Cavendish Grand Nain’.

TABLE 2 Resistance of Plants to Fusarium wilt tropical race 4 (TR4).Number Number Percent Variety of Plants Infected Infected ‘CavendishGrand Nain’ 50 32 64 ‘Cavendish Williams’ 50 38 76 (unpatented)‘RGA2-5’(unpatented) 50 14 28 ‘RGA2-2’(unpatented) 50  8 16‘RGA2-3’(unpatented) 50  3  6 ‘QCAV-4’ 50  0  0

TABLE 3 Fruit Production Characteristics Plant crop Ratoon 1 No. of No.of Bunch fingers Bunch fingers weight (top Cycle 1 weight (top Cycle 2Line (kg) hand) (days) (kg) hand) (days) Grand Nain 33.1 24.0 377.3 29.920.0 212.4 Williams 30.7 23.0 324.7 29.8 22.2 212.9 RGA2-2 29.1 22.2268.3 27.6 22.3 223.8 RGA2-3 30.1 24.9 328.9 23.8 20.2 200.8 _QCAV-428.1 22.5 331.1 24.3 19.7 199.2 RGA2-5 35.5 24.8 341.6 30.6 22.0 212.9Ratoon 2 Bunch weight No. of fingers Line (kg) (top hand) Cycle 3 (days)Grand Nain 31.7 26.5 206.6 Williams 40.4 25.9 211.8 RGA2-2 28.1 24.1215.5 RGA2-3 29.2 26.1 211.7 _QCAV-4 33.7 25.3 206.9 RGA2-5 37.3 29.5212.8

Height of about 180 to 250 cm—shorter than Giant Cavendish and tallerthan Dwarf Cavendish cultivars.

Moderate adult pseudostem width.

Relatively large bunch size.

Moderate fruit size.

Solid green leaf colour.

TABLE 4 Additional phenotypic details for ‘QCAV-4’* CharacteristicQCAV-4 phenotype Height of the pseudostem 2 - (2.1 to 2.9 m). (meanvalue = 2.78 m for three plants). Leaf habit/growth habit (upright, 3 -Drooping. spreading, drooping) Pseudostem diameter 83.7 cm (mean valuefor 3 plants). Attitude of petiole wings at base 1 - Open with marginsspreading. (curved outwards, straight, slightly curved inwards,moderately curved inwards, overlapping) Petiole margins 5 - Not wingedand not clasping the pseudostem. Petiole wing type 1 - Dry. Edge ofpetiole margin color 1 - Colourless (without a colour line along)Petiole length 1 - ≤50 cm. Typically measured at 38 cm. Blotches at thepetiole base 3 - Large blotches Color designation of leaf blade Green(137C) midrib on lower side Leaf blade shape of base (both sides 1 -Both sides rounded. rounded, one side rounded and one side acute, bothsides acute) Waxiness of lower side of leaf blade 3 - Moderately waxy.Leaf blade length 3 - 221 to 260 cm. Typically measured at 222.7 cm.Leaf blade width 3 - 81 to 90 cm. Typically measured at 90 cm. Leafblade ratio length/width 5 - 2.4 to 2.6. Typically measured at 2.5.Appearance of leaf lower surface 1 - Dull. Color designation of midribGreen (137C) ventral surface Peduncle length, width, diameter 2 - 31-60cm 2 - 7-12 cm Empty nodes on peduncle 3-4 Peduncle hairiness 3 - Veryhairy, short hairs (similar to velvet touch). Bunch position 1 - Hangingvertically. Bunch shape (cylindrical, irregular, 1 - Cylindrical.conical) Bunch appearance 1 - Lax (one can easily place one's handbetween the hands of fruit). Rachis type 2 - Present and male bud may bedegenerated or persistent. Rachis position 1 - Falling vertically. Malebud type 1 - Normal (present) Male bud shape 3 - Intermediate. Bractbase shape 1 - Small shoulder. Bract apex shape 2 - Slightly pointed.Color of the bract internal face 169A. Prominence of scars on the rachis2 - Not prominent. (weak, strong) Fading of color on bract base 1 -Color discontinuing towards the base (loss of pigmentation at the base).Male bract shape 3 - Ovate. Typical width of bract Approximately 12 cmTypical length of bract Approximately 28 cm Male bract lifting 3 -Lifting two or more at a time. Bract behavior before falling 1 -Revolute (rolling). Wax on the bract 3 - Moderately waxy. Presence ofgrooves on the bract 2 - Moderate grooving (parallel ridges aredistinguishable). Male flower behavior 4 - Neutral/male flowerspersistent. Persistence of bracts on the rachis 3 - Male flowers/bractsabove the (absent or weak, strong) male bud (but the stalk is bare aboveflowers/bracts). Shape of bract apex (narrow acute, 2 - Slightlypointed. broad acute, right angle, obtuse, emarginate) Fruit position3 - Curved upward (obliquely, at a 45° angle upward). Fruit shapelongitudinal curvature 3 - Curved (sharp curve) Fruit longitudinalridges (absent or 2 - Slightly ridged weak, moderate, strong) Fruitshape of apex 3 - Blunt-tipped (rounded, truncate, bottle-necked,pointed) Fruit persistence of floral organs 2 - Persistent style.Adherence of the fruit peel 1 - Fruit peels easily Cracks in fruit peel1 - Without cracks Fruit eating quality and main use 1 - Dessert*phenotypic characteristics are presented using the descriptors set outin IPGRI, I. MAD. 1996. Descriptors for Banana. For most, observationsfrom 3-6 individual plants.

TABLE 5 ‘QCAV-4’ bunch characteristics* BUNCH CHARACTERISTICS DIA- PLANTLENGTH METER ATTITUDE ID (cm) (cm) SHAPE OF FRUIT 20221 88.3 122.6CONICAL MODERATLY TURNED UP 20223 72.0 123.0 CONICAL MODERATLY TURNED UP20229 83.0 127.0 CONICAL MODERATLY TURNED UP 20231 75.0 113.0 CONICALMODERATLY TURNED UP 20241 72.0 120.0 CONICAL MODERATLY TURNED UP 2024277.5 110.0 CONICAL MODERATLY TURNED UP 20247 76.0 132.0 CONICALMODERATLY TURNED UP 20248 82.0 142.0 CONICAL MODERATLY TURNED UP 2025188.0 122.5 CONICAL MODERATLY TURNED UP 20255 59.0 118.0 CONICALMODERATLY TURNED UP 20257 88.5 122.3 CONICAL MODERATLY TURNED UP 2026383.0 128.0 CONICAL MODERATLY TURNED UP 20268 80.0 146.0 CONICALMODERATLY TURNED UP 20269 88.5 126.0 CONICAL MODERATLY TURNED UP 2027384.0 120.0 CONICAL MODERATLY TURNED UP BUNCH CHARACTERISTICS COM- Ripen-LONGI- PLANT PACT- # OF ing CURVA- TUDINAL LENGTH ID NESS HANDS stageTURE RIDGES (cm) 20221 MEDIUM 11 6 EVENLY MODER- 16.0 CURVED ATE 20223MEDIUM 8 6 EVENLY MODER- 14.6 CURVED ATE 20229 MEDIUM 10 6 EVENLY MODER-14.4 CURVED ATE 20231 MEDIUM 10 6 EVENLY MODER- 14.2 CURVED ATE 20241MEDIUM 6 EVENLY MODER- 13.6 CURVED ATE 20242 MEDIUM 10 6 EVENLY MODER-14.0 CURVED ATE 20247 MEDIUM 10 6 EVENLY MODER- 13.8 CURVED ATE 20248MEDIUM 10 6 EVENLY MODER- 15.4 CURVED ATE 20251 MEDIUM 11 6 EVENLYMODER- 15.4 CURVED ATE 20255 MEDIUM 8 6 EVENLY MODER- 13.7 CURVED ATE20257 MEDIUM 10 6 EVENLY MODER- 14.5 CURVED ATE 20263 MEDIUM 10 6 EVENLYMODER- 13.8 CURVED ATE 20268 MEDIUM 9 6 EVENLY MODER- 15.5 CURVED ATE20269 MEDIUM 11 6 EVENLY MODER- 14.0 CURVED ATE 20273 MEDIUM 11 6 EVENLYMODER- 13.0 CURVED ATE *Observations from 15 individual plants. Bunchaverages:_Length: 79.8 cm; Diameter: 124.8 cm; No. hands: 9.9

TABLE 6 ‘QCAV-4’ fruit characteristics FRUIT CHARACTERISTICS THICK-LENGTH NESS FRUIT OF OF COLOUR PLANT WIDTH PEDICEL SHAPE OF PEEL OF PEELID (cm) (cm) APEX (mm) (RH52015) 20221 3.5 2.8 TRUNCATE 3.8 LightGreenish Yellow 3B- 3D and 4B 20223 3.2 3.4 TRUNCATE 4.1 Light GreenishYellow 3B- 3D and 4B 20229 3.4 2.9 TRUNCATE 3.2 Light Greenish Yellow3B- 3D and 4B 20231 3.4 3.0 TRUNCATE 3.2 Light Greenish Yellow 3B- 3Dand 4B 20241 3.4 3.2 TRUNCATE 3.3 Light Greenish Yellow 3B- 3D and 4B20242 3.5 2.6 TRUNCATE 3.0 Light Greenish Yellow 3B- 3D and 4B 20247 3.33.4 TRUNCATE 3.5 Light Greenish Yellow 3B- 3D and 4B 20248 3.9 3.3TRUNCATE 3.5 Light Greenish Yellow 3B- 3D and 4B 20251 3.3 3.3 TRUNCATE3.7 Light Greenish Yellow 3B- 3D and 4B 20255 3.4 2.2 TRUNCATE 3.2 LightGreenish Yellow 3B- 3D and 4B 20257 3.6 2.5 TRUNCATE 4.0 Light GreenishYellow 3B- 3D and 4B 20263 3.3 3.2 TRUNCATE 4.5 Light Greenish Yellow3B- 3D and 4B 3.7 3.2 TRUNCATE 3.1 Light 20268 Yellow 3B- 3D and 4B20269 3.4 3.8 TRUNCATE 3.2 Light Greenish Yellow 3B- 3D and 4B 20273 2.83.1 TRUNCATE 2.7 Light Greenish Yellow 3B- 3D and 4B FRUITCHARACTERISTICS PLANT ADHERENCE COLOUR OF FIRMNESS PRESENCE ID OF PEELFLESH OF FLESH OF SEED 20221 PEELS CREAM MEDIUM ABSENT EASILY 20223PEELS CREAM MEDIUM ABSENT EASILY 20229 PEELS CREAM MEDIUM ABSENT EASILY20231 PEELS CREAM MEDIUM ABSENT EASILY 20241 PEELS CREAM MEDIUM ABSENTEASILY 20242 PEELS CREAM MEDIUM ABSENT EASILY 20247 PEELS CREAM MEDIUMABSENT EASILY 20248 PEELS CREAM MEDIUM ABSENT EASILY 20251 PEELS CREAMMEDIUM ABSENT EASILY 20255 PEELS CREAM MEDIUM ABSENT EASILY 20257 PEELSCREAM MEDIUM ABSENT EASILY 20263 PEELS CREAM MEDIUM ABSENT EASILY PEELSCREAM MEDIUM ABSENT EASILY 20269 PEELS CREAM MEDIUM ABSENT EASILY 20273PEELS CREAM MEDIUM ABSENT EASILY *Observations from 15 individualplants. Fruit averages: _Length: 14.4 cm; Width: 3.4 cm; Peel thickness:0.35 cm

TABLE 7 Average cycle time in days per cycle for ‘QCAV-4’ healthy plantsonly* Cycle time days Plant R1 R2 R3 R4 ‘RGA2-2’ 339.5 223.8 208.1 216219.4 ‘RGA2-3’ 329 200.8 210.7 201.5 187 ‘QCAV-4’ 331.1 199.2 210.8174.9 189.8 ‘RGA2-5’ 341.6 212.9 208.6 205.4 207.1 ‘Grand Nain’ 327.3211.7 206.4 206.2 213.5 ‘Williams’ 324.7 212.3 219.3 206.3 232*Observations from 50 individual plants for each variety.

TABLE 8 Average yield in kg per cycle for ‘QCAV-4’ healthy plants only*Average yield Kg Plant R1 R2 R3 R4 ‘RGA2-2’ 29.1 27.6 28 31.1 36.4‘RGA2-3’ 30 23.8 27.8 25.4 31.9 ‘QCAV-4’ 28.1 24.3 31.7 28.5 34.8‘RGA2-5’ 35.5 30.6 32.3 32 35.8 ‘Grand 33.1 29.6 32.1 29.8 35.7 Nain’‘Williams’ 30.7 30.1 35.1 33.7 37.4 *Observations from 50 individualplants for each variety.

TABLE 9 Comparative TR4 resistance* Cumulative new infections per cycle(%) Plant R1 R2 R3 R4 ‘RGA2-2’ 2 2 2 8 20 ‘RGA2-3’ 2 2 2 2 6 ‘QCAV-4 0 00 0 2 ‘RGA2-5’ 2 6 10 16 36 ‘Grand Nain’ 6 26 40 54 66 ‘Williams’ 2 1422 38 84 *Observations from 50 individual plants for each variety.Southern Analysis:

Genomic DNA was extracted from ‘QCAV-4’ and wild type (non-transformed)Cavendish Grand Nain. The DNA was digested with a restriction enzyme,electrophoresed through an agarose gel, transferred to a membrane, andprobed with a labelled RGA2 probe.

As shown in FIG. 1A, Southern analysis was consistent with fourtransgene copies in ‘QCAV-4’, in addition to the endogenous RGA2 genes.Cavendish Grand Nain is a triploid and it could be expected to havethree endogenous copies of RGA2. Two distinct bands were identified inthe wild type, indicating that two of three endogenous copies may havemigrated together.

Similar experiments were performed with wild type Cavendish Grand Nain(parent) and independent transgenic lines ‘RGA2-2’, ‘RGA2-3’, ‘RGA2-4’(clonal progenitor of ‘QCAV-4’), ‘RGA2-5’ and ‘RGA2-7’. As shown in FIG.1B, each line has a distinct pattern of transgenic RGA2 insertions, inaddition to the three endogenous RGA2 copies.

Genome sequencing:

Long Read Sequencing of Event ‘QCAV-4’

High molecular weight genomic DNA (with average fragment size >50 Kb)was isolated from young in vitro leaf tissue of ‘QCAV-4’ using GenElutePlant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA). For long-readsequencing on PacBio Sequel II platform (Novogene, China), asize-selected library with an insert size of 20 Kb was generated. Atotal of ˜75 Gbp data was obtained in CLR mode (4.9 M reads with a readlength N50 of 17,973 bp). This corresponds to ˜42× coverage of theCavendish genome at the haplotype level. SAN-3 binary vector T-DNAsequence was used to filter out long-reads from the total genomic pool.About 80 long-reads which mapped onto the T-DNA sequence were thenassembled using Flye plugin in Geneious Prime 2020. A single ˜27 kbT-DNA insertion locus was assembled. This sequence, along with 5 kbflanking sequence, was polished using ‘RGA2-4’ genomic Illumina shortreads (previously generated using Novaseq 6000) to correct a few Flyeassembly errors (short indels). Nucleotide BLAST using the two flankingsequences of this T-DNA locus revealed that the insertion of T-DNA locushas occurred in chromosome 6 of the banana genome.

Details of the T-DNA Insertion

Event ‘QCAV-4’ contains a complex T-DNAs insert of 26,849 bp at a singlegenomic location on chromosome 6 between position 29,939,311 and29,939,427 (−strand) creating a 116 bp deletion. The insert is locatedin an intergenic region between two intact predicted genes:Ma06_t28200.1 (a putative Malectin_like domain-containing protein) atposition chr06:29,931,700..29,937,001 (+strand) and Ma06_t28210.1 (amalectin_like domain-containing protein) at positionchr06:29,944,119..29,947,729 (+strand). Both genes are not affected bythe insertion and it is not predicted that the insertion will affecttheir expression.

The insert itself is composed of three full and functional copies of the6702 bp T-DNA (T-DNA 1 to 3, see FIGS. 2A-2C). In addition, twofragmented portions of the ‘RGA2’ ORF have recombined in oppositedirections and inserted between T-DNA2 and T-DNA3. There are twogenome/T-DNA and 3 inter T-DNA junctions with various levels ofrearrangement (FIGS. 2A-2C).

New Open Reading Frames (ORFs) Analysis

The analysis identified 7 new ORFs (SEQ ID NOS: 1-7), all originatingfrom these rearranged genome/T-DNA and inter T-DNA junctions. New ORFsAA sequences as follows:

>ORF_151_(frame_2)  (SEQ ID NO: 1)MWVCVSDDFDVKRITREITEYATNGRFMDLTNLNMLQVNLKEEIRGTTFL LVLDDVWNEDPVKWESLLAPLDAGGRGSVVIVTTQSKKVADVTGTMEPYV LEELTEDDSWSLIESHSFREASCSSTNPRMEEIGRKIAKKISGLPYGATA MGRYLRSKHGESSWREVLETETWEMPPAASDVLSALRRSYDNLPPQLKLC FAFCALFTKGYRFRKDTLIHMWIAQNLIQSTESKRSEDMAEECFDDLVCR FFFRYSWGNYVMNDSVHDLARWVSLDEYFRADEDSPLHISKPIRHLSWCS ERITNVLEDNNTGGDAVNPLSSLRTLLFLGQSEFRSYHLLDRMFRMLSRI RVLDFSNCVIRNLPSSVGNLKHLRYLGLSNTRIQRLPESVTRLCLLQTLLLEGCELCRLPRSMSRLVKLRQLKANPDVIADIAKVGRLIELQELKAYNVD KKKGHGIAELSAMNQLHGDLSIRNLQNVEKTRESRKARLDEKQKLKLLDL RWADGRGAGECDRDRKVLKGLRPHPNLRELSIKYYGGTSSPSWMTDQYLP NMETIRLRSCARLTELPCLGQLHILRHLHIDGMSQVRQINLQFYGTGEVS GFPLLELLNIRRMPSLEEWSEPRRNCCYFPRLHKLLIEDCPRLRNLPSLP PTLEELRISRTGLVDLPGFHGNGDVTTNVSLSSLHVSECRELRSLSEGLL QHNLVALKTAAFTDCDSLEFLPAEGFRTAISLESLIMTNCPLPCSFLLPS SLEHLKLQPCLYPNNNEDSLSTCFENLTSLSFLDIKDCPNLSSFPPGPLC QLSALQHLSLVNCQRLQSIGFQALTSLESLTIQNCPRLTMSHSLVEVNNSSDTGLAFNITRWMRRRTGDDGLMLRHRAQNDSFFGGLLQHLTFLQFLKIC QCPQLVTFTGEEEEKWRNLTSLQILHIVDCPNLEVLPANLQSLCSLSTLY IVRCPRIHAFPPGGVSMSLAHLVIHECPQLCQHVPGTFGHP*   >ORF_156_(frame_3) (SEQ ID NO: 2)MRFLPEVSACPWHIWSSMNALSCVSMSLAHLVIHECPQP*   >ORF_111_(frame_1) (SEQ ID NO: 3)MHVMLYSWIRRGREDDSGGSIRITHYYGQFKLKAGANSH*   >ORF_87_(frame_1) (SEQ ID NO: 4) MCYSDRSSRVVFPAPPNPTIEHHMHSGIIENKNLKFSTEKCFVIVRRLVH KTENVK*   >ORF_71_(frame_3)  (SEQ ID NO: 5)MPLPTVVPKMDPHPRGASWKKKTFQPRLQSKWIDVNMLEQLWRIYCGVNK LTLRQLNNTLRTFLMY*   >ORF_49_(frame_3)  (SEQ ID NO: 6)MTKCARDMLTQLRAFMDDQMCQGHADTSGRKRMDSWASDDVQGAEGAEAL QVCRQYLQVWTINDVQNLKRSKVSPLLFLFAGEGYELWTLADL*   >ORF_7_(frame_2) (SEQ ID NO: 7) MDRHLKSRIRFWFKQQWPRQLNNTLRCKQIDA*  

Assessment of the Expression of the Seven New ORFs

To assess the expression potential of the seven newly identified ORFs,two RNAseq Illumina libraries were used. Root and leaf RNAseq librariescontaining 274,556,348 and 268,119,840 reads, respectively were mappedto the reconstructed insertion locus. From this analysis, 1,029,853 and781,191 reads originating from the leaf and root RNAseq datasetrespectively mapped to the insert sequence. No read from either librarymapped continuously across any of the seven newly identified ORFs,confirming the lack of mRNA originating from them in event QCAV-4.

Bioinformatic Assessment of the Allergenicity Potential the Seven NewORFs

In silico analyses performed (see below) to compare amino acid sequenceof each new ORF to known allergenic proteins in the Food AllergyResearch and Resource Program (FARRP) dataset, which is availablethrough AllergenOnline (University of Nebraska). Full length sequence (Evalue <10⁻⁵), 80-mer sliding window (35% homology with E value <10⁻⁴)and 8-mer exact match searches identified no sequences similaritybetween any of the 7 new ORFs and known allergens in the database.

Bioinformatic Assessment of the Toxicity Potential of the Seven New ORFs

Potential structural similarities shared between the seven new ORFs andsequences in a protein toxin database were evaluated using the BasicLocal Alignment Search Tool (BLAST) available within the Geneiousprogram.

A blastp search using the BLOSUM45 similarity scoring matrix and theamino acid sequence from the seven new ORFs as the query sequence didnot return any accessions of biological significance from the toxindatabase with an E-score acceptance criteria lower than 1×10⁻⁴.

We claim:
 1. A new and distinct variety of banana plant, substantiallyas herein shown and described.